Vitamin D deficiency diseases are characterized by defective bone formation. With the identification of the major pathways and products of the metabolism of vitamin D, there has followed an intense effort to determine the role of vitamin D metabolites in the development and remodelling of the skeleton. Yet two major questions remain at the center of controversy regarding the role of vitamin D in skeletal homeostasis. Does 1,25(OH)2D3 have direct anabolic effects on bone forming cells and are there any actions of metabolites other than 1,25(OH)2D3 which are physiological significance to the formation and/or mineralization of bone matrix? The studies proposed are designed to examine the regulation by vitamin D metabolites of two osteoblast functions which are associated with bone formation, alkaline phosphatase and collagen synthesis. Primary cultures of cells of the osteoblast line isolated from neonatal rat calvaria will be used to test the hypothesis that one or more metabolites of vitamin D have anabolic effects on bone formation and that 1,25(OH)D3 and 24,25(OH)2D3 have qualitatively different regulatory functions in bone forming cells. We will attempt to determine the mechanism whereby vitamin D metabolites modulate bone cell alkaline phosphatase activity with respect to synthesis, degradation and secretion of the enzyme. The effects of these vitamin D metabolites on collagen synthesis will be examined under conditions in which alkaline phosphatase activity is modulated to determine if the regulation of these two cell functions can be correlated. Comparative studies will be carried out in serum-free cultures of chick embryo calvaria cells and ROS 17/2.8, and osteognic sarcoma clonal cell line.